Human multipotent stromal cells or mesenchymal stem cells (MSCs), capable of self renewal and differentiating into various mesenchymal tissues (Prockop, Science 276, 71-74, 1997), have emerged as a promising tool for clinical applications in, for example, cell-based therapy for osteogenesis imperfecta (Horwitz et al., Nat Med 5, 309-313, 1999) and tissue engineering in cartilage and bone (Caplan, Tissue Eng 11, 1198-1211, 2005). MSCs are also applied for cardiac therapeutics by preventing deleterious remodeling and improving recovery (Pittenger and Martin, Circ Res 95, 9-20, 2004). However, the variations in the isolation techniques, growth media, and culture conditions cause a remarkable difference in their proliferation and differentiation capacity (Pittenger, Methods Mol Biol 449, 27-44, 2008). Further, many studies have consistently noticed a senescent tendency of MSCs upon expansion (Bonab et al., BMC Cell Biol 7, 14, 2006; Shibata et al., Stem Cells 25, 2371-2382, 2007; and Wagner et al., PLoS ONE 3, e2213, 2008). Thus, the difference in stem cell properties and the senescence encountered during expansion hinder the clinical applications of MSCs.
Hypoxia has been known to regulate several cellular processes and signal transductions by the expression of Hypoxia Inducible Factor-1 (HIF-1), a heterodimer consisting of the constitutively expressed aryl hydrocarbon receptor nuclear translocator (ARNT) and the hypoxic response factor HIF-1α. HIF-1α is regulated by the cellular O2 concentration and determines the transcriptional activity of HIF-1 (Semenza, Curr Opin Genet Dev 8, 588-594, 1998). Most of the effects of HIF-1α were investigated on cancer cells. HIF-1α, induced during ischemia, environments in the tumour nature course and after treatment, stimulates proliferation (Stoeltzing et al., J Natl Cancer Inst 96, 946-956, 2004), inhibits apoptosis (Akakura et al., Cancer Res 61, 6548-6554, 2001), induces VEGF expression and angiogenesis (Stoeltzing et al., J Natl Cancer Inst 96, 946-956, 2004), and promotes tumour progression and metastasis (Zhou et al., Cancer Lett 237, 10-21, 2006). Low-density culture improves the efficiency of expansion at the earliest passages (see, U.S. Pat. No. 7,374,937). As described above, MSCs enriched by low-density culture method also undergo senescence and lost stem cell properties (also see Table 1). Since bone marrow, the original environment of MSCs, is hypoxic with the oxygen tension around 1 to 7% (Harrison, J. S. et al., Blood 99, 394, 2002), the inventors of present invention hypothesize that hypoxic culture provides more benefits than normoxic culture. Although hypoxic condition was used in the expansion of neural stem cell previously (see, US20070264712 A1), low-density culture combined with hypoxic culture has not yet been used in expanding MSCs.
Twist, a helix box transcription factor, has been known to promote tumour metastasis by inducing epithelial-mesenchymal transition (EMT) (Yang et al., Cell 117, 927-939, 2004). Twist cooperates with N-myc to induce tumouriogenic transformation (Valsesia-Wittmann et al., Cancer Cell 6, 625-630, 2004). Twist and Snail, another inducer of EMT, were proved to increase cells with cancer stem cell properties when overexpressed in breast cancer cells (Mani et al., Cell 133, 704-715, 2008). Further, Twist can overcome oncogene-induced senescence to complete oncogenic transformation (Ansieau et al., Cancer Cell 14, 79-89, 2008). Recently, the HIF-Twist axis has been proved in head and neck cancer and is involved in tumour metastasis (Yang et al., Nat Cell Biol 10, 295-305, 2008). Stem cells and cancer cells share a lot of similarities in gene expression, cellular processes, signal transductions, however there are few, if any, studies research the effects of HIF-Twist on stem cells.
The results of the invention provide evidences for proposing a general protocol for rapid and efficient expansion of MSCs by combining low density culture and hypoxic culture. Inventors found hypoxic culture not only prevented senescence that was noted in expanded MSCs but also increased embryonic gene expression and differentiation efficiency. The underlying mechanism mediating the increase in stemness by hypoxic culture on MSCs occurred through downregulation of E2A-p21 by the HIF-Twist pathway.